JPH03158168A - Blood component separating method and blood component separator - Google Patents

Abstract

PURPOSE:To provide a method and a device to remove mixed leucocyte with high efficiency by using a blood formulation consisting mainly of erythrocyte. CONSTITUTION:A blood component separator is formed such that a container is filled with a very fine fibre laminate having an average diameter of 0.4-1.5mum so that bulk density is 0.1-0.4g/cm<3>, and a thickness is 3-10mm. Whole blood after sampling of which 24 hours elapse or a blood formulation prepared by the whole blood is caused to pass the separator. During filling with a fibre laminate, the more bulk density is higher, the more leucocyte removing capacity is increased, it is imperative that bulk density is 0.1-0.4 g/cm<3>, and it is prefer ably 0.15-0.3g/cm<3>.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Field of Application) The present invention relates to a blood component separation method and a blood component separator. Specifically, the present invention relates to a method and apparatus for removing contaminated leukocytes at a high rate from blood products for transfusion, especially blood products whose main constituents are red blood cells.

(Prior Art) In recent years, in the field of blood transfusion, component transfusion, which provides only the components required by a patient, has become preferred in place of conventional whole blood transfusion.

It has become clear that the conventional transfusion of whole blood or concentrated red blood cells causes various side effects because white blood cells, platelets, plasma, etc. mixed in blood products are also transfused at the same time. Side effects range from relatively minor ones such as headache, fever, nausea, and nausea, to GV HD after blood transfusion,
There are some serious effects on patients, such as alloantigen sensitization and viral infection, but these are mainly caused by white blood cells. Therefore, it has come to be considered that it is better to transfuse red blood cell preparations from which leukocytes have been removed for general blood transfusion patients who require only red blood cells.

In order to prevent relatively minor side effects such as headache, fever, nausea, and nausea, it is necessary to remove the remaining white blood cells to below 10 to 10-2, and to prevent GVHD after blood transfusion.
Although there is no established theory for preventing alloantigen sensitization, it is effective to suppress the leukocyte residual rate to 10-3 or less.
It is thought that it can be almost completely prevented by reducing it to 0-4 or less (Clinical and Research J 86 (2): 349.198
9).

Although there is no established theory regarding viral infections, viruses that are thought to exist only in white blood cells, such as cytomegalovirus and adult TNfJ cell leukemia virus, are removed to a white blood cell residual rate of 10-4 to 10-6 or less. It is hoped that this will prevent the infection.

Methods for removing white blood cells from blood products can be broadly divided into two methods: a method that uses a centrifuge to separate red blood cells and white blood cells by taking advantage of the difference in specific gravity, and a filter method that uses fiber materials or sponge-like structures as filter media. There are two types: filter method,
In particular, the method of adsorbing and removing leukocytes using a nonwoven fabric is widely used because it has advantages such as excellent leukocyte removal ability, simple operation, and low cost. However, the leukocyte removal ability of these nonwoven fabric filters disclosed in JP-A-60-193468, JP-A-63-175157, JP-A-63-264073, etc. has a leukocyte removal rate of 100 in some cases. Other than those listed as %, all have a white blood cell residual rate of 10'''3 or higher, and can only prevent relatively minor side effects such as headache, fever, nausea, and side effects derived from white blood cells. In addition, even when it is stated that the leukocyte removal rate is 100%, the leukocyte measurement technology that existed at the time was used, i.e., the white blood cell concentration was measured using a hemocytometer after diluting the blood with Turk's solution etc. However, considering the fact that the sensitivity of white blood cell measurement was around 10"3, the method of diluting blood with physiological saline, etc. and then measuring the white blood cell concentration using an automatic blood cell counter such as a Coulter counter, It is thought that the residual rate was not 0, but 10-3 to 10"'. In fact, using the recently developed leukocyte measurement method with a measurement sensitivity of 10-7, the leukocyte removal rate was 100%.
When we tried the example described as follows, we found that the leukocyte survival rate (
10-3,/l).

The current measurement method uses a hemocytometer, but the conventional method involves diluting blood 5 to 10 times and then injecting it into the hemocytometer and counting the white blood cells contained in 4 to 8 large sections. Unlike the conventional method, only leukocytes in the blood were concentrated approximately 600 times using Ficoll, and then injected into a hemocytometer, and the leukocytes contained in 108 large sections were counted. Therefore, the measurement sensitivity is approximately 4X10'~ compared to the conventional method.
It has improved by about 10', and it is now possible to measure the survival rate to 10-7 or less.

(Problems to be Solved by the Invention) The present invention achieves a leukocyte survival rate of 10-6 or less, which can almost completely prevent side effects originating from leukocytes, and further reduces the pressure loss during blood processing by reducing hemolysis of red blood cells. range, i.e. 30
It is an object of the present invention to provide a blood component separation device and method that can complete the treatment within 30 minutes while maintaining the treatment range at a head difference of 0 mmHg or less, preferably about 1.5 m, that is, 126 m'lTlHg or less. It is something.

(Means for solving the problems) The gist of the present invention is to
! The I fiber laminate has a bulk density of 0.1 to 0.4 g7cm3. A blood component separation device comprising a container filled to a thickness of 3 to 10 mm, and blood collected within 24 hours after blood collection or blood products prepared from the blood passed through the separator. There is a method for separating blood components.

The fiber laminate in the present invention requires that the fibers constituting it have an average diameter of 0.4 to 1.5 μm,
Preferably 04~1.2μm, more preferably 094~
It is 1.0 μm. Within this range, the smaller the fiber diameter, the higher the leukocyte removal ability. This is the same - the same in potted plants -
When filling a car's worth of fiber laminate, that is, when the bulk density is constant, the thinner the fiber diameter, the greater the fiber length per unit volume of the ya fiber laminate, and in proportion to this, the This is thought to be due to an increase in the number of locations where the two are intertwined. That is, when the state of the leukocytes attached to the fiber laminate was observed using a scanning electron microscope, it was found that many leukocytes were attached near the intertwining points between the fibers. White blood cells are in contact with two or more fibers near the intertwining point, and by coming into contact with multiple fibers at the same time, the binding force is stronger than if they were in contact at a single point. It is thought that the leukocytes are able to remain in place, ie, adhere, against the force that tends to sweep away the leukocytes caused by the flow of the blood product.

Therefore, in terms of being able to provide more such fields, it is better for the diameter of the fiber 1a to be smaller. The intertwining point mentioned here is a place where leukocytes can come into contact at two or more places at the same time, and even if the fibers are not in complete contact, the distance between the fibers is relative to the diameter of the leukocyte. If the fibers are small, even if there are gaps between the fibers, they can essentially be considered as intertwining points. Fiber diameter is 1°5μm
If it exceeds , the thickness of the fiber laminate 'h1 to provide the necessary intertwining points becomes extremely large, and the volume of the blood component separator also increases proportionately, which is beyond the practical range. . On the other hand, fiber laminates with a diameter of less than 0.4 μm are extremely difficult to manufacture and are not practical.

In addition, the average diameter of fibers in the present invention refers to a value determined according to the following procedure. That is, a part of the fiber laminate is sampled and photographed using a scanning electron microscope or the like. On top of the photo, one side or a few μm based on the magnification of the photo.
A grid corresponding to about m to 10 μm is placed on the fiber, and the diameter of the fiber at each grid point of this grid is measured. Here, the diameter refers to the width of the fiber in the direction perpendicular to the fiber axis. The average diameter is the sum of the diameters measured at each grid point on the photograph divided by the number of grid points. However, if there are no fibers at the lattice points,
In the case where multiple fibers are wrapped around each other in the vicinity of the lattice point, and some of the fibers at the lattice point are shaded by other m'M1 and its width cannot be measured, Thick Na by melting! If the fiber diameter is 1I, if there are fibers with clearly different fiber diameters, etc., these data will be deleted. By the above method, the average diameter is determined from data of 20 or more grid points, preferably north of 100 grid points, and more preferably 1000 or more grid points.

Similarly, in the sense that more intertwining points can be provided, when filling a fiber laminate, the higher the bulk density, the higher the leukocyte removal ability, and the bulk density in the present invention is 0.1 to 0.
．． It is necessary that it is 4g/m', preferably 0.1
It is 5 to 0.3 g/cm3.

This conclusion was reached by the present inventors based on an unexpected fact discovered in the course of a detailed study of the causal relationship between bulk density and leukocyte removal ability.

That is, when filling a container with a fiber laminate having the same weight and fiber diameter, the more the fiber laminate is compressed in the flow direction of the blood product, that is, the thinner the thickness in the flow direction, the higher the bulk density. It was found that the leukocyte removal ability increases as the amount increases. The fiber laminate is arranged in a container in such a way that the fibers are stacked on a plane perpendicular to the flow direction of the blood product. The stiff component acts to increase the bulk of the fiber laminate, and acts as an inhibitory factor for the formation of entangled points because it increases the distance between fibers in the direction parallel to the flow direction of blood products. do. However, when the fiber laminate is compressed, the components are forced to align closer to the direction perpendicular to the flow of the blood product, compared to before compression.
As the fiber component in the direction perpendicular to the flow direction increases,
This is thought to be due to the fact that as a result of reducing the distance between the fibers in the machine direction, the fibers become closer together and the number of intertwining points increases. However, this increase in the ability to remove leukocytes per unit of melon through compression is an effect that is found only in a specific range of bulk density, and the range differs depending on the fiber diameter. For example, when a polyester nonwoven fabric produced by the melt blow method is used in a fiber laminate, the bulk density is 0.1 g /
The effect appears when cm is 3 or more 1, similarly 0.96μ
0.16g/cm' or more for m, 0.2 for 1.2μm
The effect appears at 3 g/cm3 or more, but 1.
At 7 μm, such an effect could no longer be found even at 0.4 g/cm'. At a bulk density of less than 0.1 g/cm3, where such an effect is not found, the leukocyte removal ability is insufficient because the number of confounding points that can be provided is insufficient. on the other hand,
When it exceeds 0.4 g/cm3, although there are many intertwined points,
Since the porosity decreases, the passage resistance of red blood cells increases, and it is no longer possible to secure the necessary flow rate within a practical pressure drop.

The above facts are clearly different from the conventional idea that the ability to remove leukocytes is determined by the surface area of the fiber, and were discovered based on the number of confounding points, and provide a new perspective. Taking this perspective further, it is assumed that all of the constituent fibers in the fiber laminate lie in a plane perpendicular to the flow direction of the blood product, and that the distance between the fibers in the flow direction is the diameter of a white blood cell. It is most preferable that it be arranged so that it is smaller than . In other words, it is preferable that the laminated state of the fibers exhibits excellent anisotropy in the flow direction of the blood product and in the direction perpendicular to the flow direction.

As a means of increasing the anisotropy, there are methods such as compressing the fiber laminate and placing it in a container as described above, and adjusting the manufacturing conditions of the fiber laminate so that it has a relatively small basis weight and a small bulk. be.

Further, the fiber laminate in the present invention needs to have a thickness of 3 to 10 mm in a direction parallel to the flow direction of the blood product, and more preferably 5 to 10 mm.

As a result of a detailed study by the present inventors on the relationship between the thickness of a 1-ft fiber laminate and the change in leukocyte concentration when passing through the fiber laminate, the leukocyte concentration was found to be an exponential function with respect to the thickness of the fiber laminate. It was found that this phenomenon can be understood as an adsorption phenomenon that can be organized using two parameters: the number of collisions and the probability of adsorption per collision. That is, as the white blood cells pass through the fiber laminate, they repeatedly come into contact with the intertwined points, adhere at a certain probability per contact, and gradually decrease their concentration until they reach the 8th side of the fiber laminate. You can think of it as heading towards . For this reason, the white blood cell concentration ratio before and after passage, that is, the white blood cell residual rate, is
Decrease exponentially. Therefore, when taking the logarithm of the leukocyte survival rate, this value decreases linearly with the thickness of the fiber laminate. It has been empirically known that the greater the thickness, the better the ability to remove leukocytes, but this is the first time in the present invention that this has been quantitatively understood. However, surprisingly, it became clear that from around the time when the leukocyte survival rate reached 10-4 or less, the slope of the logarithmic straight line of the leukocyte survival rate no longer maintained its initial slope, but became gentler. became. In other words, this suggests that there is a group of white blood cells that are difficult to adhere to. This phenomenon was discovered for the first time by using a highly sensitive white blood cell counting method, and could not have been predicted at all from the knowledge of conventional counting methods, which had a sensitivity of about 10-3. It is something that

In order to achieve the leukocyte survival rate of 10-6, which is the objective of the present invention, it is important to determine how efficiently these leukocytes with low adhesion can be removed.

Therefore, it is necessary to make the thickness of the fiber laminate in the direction parallel to the flow direction of the blood product considerably larger than the thickness expected from the initial inclination. Specifically, it must be 3 mm or more, and 5 mm or more. It is preferable to take the above. On the other hand, if the thickness exceeds 10 mm, it is possible to achieve 10-6 even if the average fiber diameter and bulk density are out of the range of the present invention, but the pressure Wi will increase extremely and the briming volume will become large. Too much will make it impractical.

The requirements for fiber diameter, density, and thickness (as described above) for the M fiber laminate cannot be specified independently, but have a unique range depending on the combination thereof. For example, the fiber diameter is 0. 5μm, bulk density 0.15g
/cm3. Performance equivalent to J9 7.8mm, 0.75
μm, 0.15g/cm3.9.1mm and 1.184
m, 0.30g/am”, 9.1mm combination and 0.30g/am”.
75. jm, 0.22g/cm3, 5.3mm, and 0
, 96 μm, 0. 24g/am'9. 1m
It is possible to obtain by a combination of m.

Examples of the fiber laminate in the present invention include nonwoven fabrics produced by melt blowing, flash spinning, papermaking, etc., as well as paper, woven fabric, mesh, and the like. Examples of fiber materials include synthetic fibers such as polyamide, aromatic polyamide, polyester, polyacrylonitrile, polytrifluorochloroethylene, polymethyl methacrylate, polystyrene, polyethylene, and polypropylene, cellulose, and cellulose acetate.・Regenerated fibers such as

In addition, the blood products used in the present invention can be used within 24 hours after blood collection.
It must be prepared from whole blood within 1 hour, or after removing part of the plasma components, platelet-rich components, buffy coat, etc. from the whole blood by centrifugation, membrane separation, etc. , preferably within 15 hours, more preferably within 8 hours. If it exceeds 24 hours, the ability to remove leukocytes decreases. The reason for this is not clear, but it is thought that when whole blood is stored for a long time, the adhesion of leukocytes is affected by other blood components and decreases. If a red blood cell preparation is prepared after removing plasma components, platelet-rich components, puffyco-1, etc. by normal centrifugation or membrane separation within 24 hours of blood collection, the storage time is long. Although no problem will arise in terms of leukocyte removal performance, it is preferable to do so within 4 days after blood collection. In addition, the blood product hematoxot is
It is preferably 40 to 70%, more preferably 50 to 55%.
% is more preferable. The blood products used in the present invention preferably have red blood cells as a main component, but other products such as platelet concentrates can also be used.

(Example) Next, the present invention will be explained in more detail with reference to Examples.

(Example 1) Average diameter 0.75 μm manufactured by melt blowing method
A fibrous laminate made of polyester nonwoven fabric of 0.25 mm was placed in a container with an effective filtration cross section of 67 mm x 67 mm, with a bulk density of 0. t80g
/cm3 and a thickness of 9.1 mm to prepare a blood component separator. 8 hours after blood collection, CPD6 was added to the separator.
After 8 hours of blood collection from 450 mu of human whole blood to which 3 m and Q had been added, 270 m
Add 80 m J2, hematocrit 54%, 35
18 hours after adjustment (26 hours after blood collection), the blood product adjusted to 0 mJl was passed through the tube at a height of 2.2 m at room temperature.
Continue to apply 70 mfl of physiological saline at a height of 2.2 m.
The passing liquid meter is 350 m, and the Q is 1000 rnJ.
2 blood bags were collected. Immediately after collection, add 5% Ficoll 400DLEBSS solution (hereinafter referred to as Ficoll solution)
The same volume as the passing fluid was added to the blood bag while thoroughly mixing, and the bag was fixed on a blood separation stand and left standing for 40 minutes. After leaving it to stand still, remove it by hand, taking care not to disturb the sedimented red blood cell layer.
After gently collecting the supernatant, Ficoll solution was again added to the blood bag with the same volume as the IF time, and the same operation was repeated.

The supernatant recovered from the two operations was transferred to Corning 253.
The mixture was dispensed into four '50 centrifuge tubes, centrifuged at 840Xg for 15 minutes, and the supernatant was discarded using an aspirator, being careful not to suck up the sediment. 200 mu in each centrifuge tube
Add 1.145% ammonium oxalate saline (hereinafter referred to as "hemolysis") and mix immediately with 468% ammonium oxalate.
The mixture was centrifuged at

Collect 4 pieces of sediment into a 15 ml centrifuge tube, add hemolysate to bring the total volume to 15 ml, let stand at room temperature for 10 minutes, centrifuge at 468Xg for 10 minutes, and collect 0.5 mu containing sediment.
The supernatant was carefully discarded. After thoroughly stirring the solution containing the sediment to obtain a single cell suspension, 69.9 mg/R acridine orange solution (staining solution) at 50 μC was added and further stirred. This liquid was added to the Barker-Turk type blood calculation board 6.
The leukocytes present in 108 large sections were counted using an epifluorescence microscope to determine the leukocyte concentration. this! Volume 0 at a time. This value was multiplied by 55 mft and further divided by 0.55 to obtain the number of passing leukocytes. 0.
The reason for dividing by 55 is that the recovery rate when collecting leukocytes using Ficoll solution is about 0.55. The leukocyte residual rate was determined by dividing the number of passing leukocytes by the number of leukocytes in the blood product determined from the leukocyte concentration in the blood product before passing through the blood component separator measured by a conventional method and the volume of the blood product. However, my IQ was -7.2. In addition, when the red blood cell recovery rate was calculated from the hemadcrit values before and after passage, it was found to be 9.
It was 5.2%. Also, after starting blood products,
The time required to finish flushing the saline was within 30 minutes.

(Example 2.3) The experiment was carried out in the same manner as in Example 1, except that the conditions of average diameter, bulk density, thickness, time to prepare blood product after blood collection, hematocrit and volume of blood product were changed. I did this. The results are shown in Table 1. The time required from the time the blood product started to flow until the physiological saline solution was completely drained was within 30 minutes in all cases.

(Leaving space below) (Examples 4 to 10) The conditions of the average diameter, LG density, paste, time to adjust the blood product after blood collection, hematocrit and volume of the blood product were changed, and the head height was 2.2 m. Except that instead of flowing at 15 mu/min using a peristaltic pump, the pressure applied to the blood component separator at the end of blood processing was measured.
The same experiment as in the example was conducted. The results are shown in Table 2.

Note that the pressure was always below 300 mmHg.

(Margins below) (Comparative Examples 1 to 4) For comparison, cases in which any of the conditions such as average diameter, bulk density, thickness, time after blood collection until preparation of blood products, etc. are outside the range specified in the present invention An experiment similar to that in the example was conducted. The results are shown in Table 3.

(Blank below) (Effects of the Invention) According to the present invention, as is clear from Tables 1 to 3, leukocytes can be removed from blood products at an extremely high rate, so the effects are remarkable.

Claims (2)

[Claims] (1) When removing contaminated leukocytes from blood products, ultrafine fiber laminates with an average diameter of 0.4 to 1.5 μm are used with a bulk density of 0.
．． After blood collection, 24 hours after blood collection, the blood was collected into a blood component separator filled in a container with 1 to 0.4 g/cm^3 and a thickness of 3 to 10 mm.
1. A method for separating blood components, which comprises passing whole blood or blood products prepared from the whole blood within an hour. (2) A blood component separator for removing contaminated white blood cells from whole blood collected within 24 hours after blood collection or blood products prepared from the whole blood, comprising a laminate of ultrafine fibers with an average diameter of 0.4 to 1.5 μm. , bulk density 0.1 to 0.4 g/cm^3, thickness 3
A blood component separator that is filled into a container to a thickness of ~10 mm.